11th International Gluten Workshop                                 Beijing, China                               August 12-...
ContentIntroductionConditions that affect reliability of gluten measurements:     • Variety of gluten-detecting antibodies...
Introduction           Coeliac disease• Coeliac disease is one of the most common food intolerances in the  world, with pr...
Introduction    Measuring gluten content• Gluten content is measured by immunological ELISA methods.• Methods are based on...
Variety of gluten-detecting antibodiesR5 antibody raised against rye secalin (Sorell et al. 1998)– currently in use    Rec...
Kanerva et al 2011 Agric Food Sci 20:206-216.11th International Gluten Workshop, Beijing, 12-15 Aug 2012Päivi Kanerva
Different reference materials• Reference material has a major influence on the quantification.• Reference material should ...
Measurement of gluten with R5 ELISA                               using different reference materialsGluten content (ppm) ...
Hydrolysis of proteins• Hydrolysis of proteins occurs during many food  manufacturing processes, e.g. brewing• Hydrolysis ...
•Rye protein hydrolysis during sourdoughprocess               Sourdough from germinated rye contained               only 2...
Protein deamidationIn addition to hydrolysis, solubility of gluten can be improved bydeamidationDeamidation improves also ...
Chemical deamidation of vital gluten  by acid treatment11th International Gluten Workshop, Beijing, 12-15 Aug 2012Päivi Ka...
Deamidation of gluten significantly Omega-gliadin sandwichreduced its immunological detection ELISAby the prolamin specifi...
ConclusionsReliability of the measurements of gluten content is questioned if thecomplexity of prolamins and changes in th...
Thank youCereal Technology Group at the University of Helsinki  Professor   Hannu Salovaara  University lecturer   Tuula S...
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How reliable are the measurements of residual gluten in gluten-free foods?

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How reliable are the measurements of residual gluten in gluten-free foods?

  1. 1. 11th International Gluten Workshop Beijing, China August 12-15, 2012How reliable are the measurements of residual gluten ingluten-free foods? Päivi Kanerva Department of Food and Environmental Sciences University of Helsinki, Finland www.helsinki.fi/yliopisto
  2. 2. ContentIntroductionConditions that affect reliability of gluten measurements: • Variety of gluten-detecting antibodies • Different reference materials • Protein hydrolysis • Protein deamidationConclusions11th International Gluten Workshop, Beijing, 12-15 Aug 2012Päivi Kanerva www.helsinki.fi/yliopisto
  3. 3. Introduction Coeliac disease• Coeliac disease is one of the most common food intolerances in the world, with prevalence of 1-2 %• Gluten proteins are known to be an environmental factor causing coeliac disease• Gluten proteins of wheat and similar proteins of barley and rye are all harmful for coeliacs• Harmful proteins trigger an adverse immunoreaction in people with coeliac disease leading to destruction of small intestine villi• Current treatment for coeliac disease is a gluten-free diet 11th International Gluten Workshop, Beijing, 12-15 Aug 2012 Päivi Kanerva www.helsinki.fi/yliopisto
  4. 4. Introduction Measuring gluten content• Gluten content is measured by immunological ELISA methods.• Methods are based on prolamin-recognizing antibodies.• The method currently recommended by Codex is known by names R5-ELISA or the method of Mendez Sandwich Competitive 11th International Gluten Workshop, Beijing, 12-15 Aug 2012 Päivi Kanerva www.helsinki.fi/yliopisto
  5. 5. Variety of gluten-detecting antibodiesR5 antibody raised against rye secalin (Sorell et al. 1998)– currently in use Recognizes prolamins of wheat, barley and rye; reacts mainly with QQPFPω-gliadin antibody raised against wheat gliadin (Skerritt&Hill 1990)– predecessor of R5 Recognizes heat-stable omega-type prolaminsα-gliadin antibody raised against wheat gliadin (Spaenij-Dekking et al. 2004) Recognizes T-cell stimulatory epitopes of wheat, barley and ryeG12 and A1 antibodies raised against 33mer (Moron et al. 2008) Recognizes prolamins of wheat, barley and rye, and weak reaction to oat prolamins; G12 reacts mainly with QPQLPY, A1 www.helsinki.fi/yliopisto with QLPYPQP
  6. 6. Kanerva et al 2011 Agric Food Sci 20:206-216.11th International Gluten Workshop, Beijing, 12-15 Aug 2012Päivi Kanerva
  7. 7. Different reference materials• Reference material has a major influence on the quantification.• Reference material should be similar to the analysed protein; however, complexicity of prolamins makes it difficult• Different standards are used today in gluten analysis: PWG gliadin other gliadin standards (e.g. Sigma) Peptide standards (synthetic or hydrolyzed) 11th International Gluten Workshop, Beijing, 12-15 Aug 2012 Päivi Kanerva www.helsinki.fi/yliopisto
  8. 8. Measurement of gluten with R5 ELISA using different reference materialsGluten content (ppm) Hordein content (ppm) Amount of barley flour (%) Amount of barley flour (%) Gliadin standard Hordein standard
  9. 9. Hydrolysis of proteins• Hydrolysis of proteins occurs during many food manufacturing processes, e.g. brewing• Hydrolysis can be enhanced by enzymes: either added or endogenous• High amounts of prolamin degrading enzymes is developed during grain germination 11th International Gluten Workshop, Beijing, 12-15 Aug 2012 Päivi Kanerva www.helsinki.fi/yliopisto
  10. 10. •Rye protein hydrolysis during sourdoughprocess Sourdough from germinated rye contained only 240-480 ppm of prolamins, which is about 0,5% from original amount Both sandwich and competitive assay gave similar results
  11. 11. Protein deamidationIn addition to hydrolysis, solubility of gluten can be improved bydeamidationDeamidation improves also foam and emulsion forming cababilities ofgluten proteinsDeamidation can be done by acid or base treatment or enzymatically Deamidation is a conversion of glutamine or asparagine to glutamic Gluten Workshop, Beijing, 12-15acid 11th International acid or aspartic Aug 2012 Päivi Kanerva www.helsinki.fi/yliopisto
  12. 12. Chemical deamidation of vital gluten by acid treatment11th International Gluten Workshop, Beijing, 12-15 Aug 2012Päivi Kanerva
  13. 13. Deamidation of gluten significantly Omega-gliadin sandwichreduced its immunological detection ELISAby the prolamin specific antibodiesR5 and omega-gliadin.R5 competitive ELISA R5 sandwich ELISA x 600 x 125 Kanerva et al. 2011. J Cereal Sci
  14. 14. ConclusionsReliability of the measurements of gluten content is questioned if thecomplexity of prolamins and changes in their structures are not carefullyconsidered.  reliability can be substantially improved by selecting right reference material  reactivity of different prolamins with different antibodies should be taken into account and results interpreted accordingly  consequences of modification to protein reactivity with antibodies should be evaluated 11th International Gluten Workshop, Beijing, 12-15 Aug 2012 Päivi Kanerva www.helsinki.fi/yliopisto
  15. 15. Thank youCereal Technology Group at the University of Helsinki Professor Hannu Salovaara University lecturer Tuula Sontag-Strohm Researchers Päivi Kanerva Xin Huang Technician Outi Brinck

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